This invention relates to materials which modulate the action of thrombin on cells of the vascular and circulating blood system. It is well known that thrombin has multiple activities. Perhaps the most well known activity of this serine protease is to convert fibrinogen to fibrin which clots blood. Additional functions of thrombin are widespread and diverse and appear to involve cellular activations which are mediated through cellular thrombin receptor(s). For example, thrombin is the most potent activator of platelets; it is chemotactic for monocytes; it is mitogenic for lymphocytes and mesenchymal cells including vascular smooth muscle cells and; it promotes numerous responses within the vascular endothelium. See, Coughlin, et al., J. Clin. Invest. 89:351-355 (1992). Because these cell activating functions of thrombin occur within the range of concentrations normally required for the clotting of blood, thrombin has been proposed to play important physiological roles not only in hemostasis and thrombosis but may also have principle roles in mediating responses to vascular injury such as leukocyte chemotaxis to mediate inflammation, cellular proliferation to mediate restenosis, glomerulonephritis, and wound repair such as occurs in bone remodeling.
The role of thrombin in acute thrombosis has been clearly established. For a review, see, Chesbro et al., Thromb. Haemostas., 66:1-5 (1991). However, thrombin's role in thrombosis is not limited to its blood clotting activity but also extends to platelet aggregation as thrombin appears to be the principle physiological mediator of platelet aggregate formation at sites of vascular injury resulting from the activation of the platelet thrombin receptor. Recent antithrombotic approaches inhibit or modulate the enzymatic activity of thrombin and include compounds such as heparin, low molecular weight heparins, PPACK, hirudins, argatroban, and the hirulogs. All of these agents inhibit the catalytic activity of the enzyme. Therefore, these agents not only inhibit the pro- and anticoagulant actions of thrombin but also the cell activating functions of thrombin as well. Accordingly, none of these agents are useful for specifically inhibiting the cellular actions of thrombin. No agent which specifically targets the thrombin receptor has been clinically developed. Previous attempts to identify a thrombin receptor inhibitor have been thwarted by the inability of researchers, until very recently, to identify the physiologically relevant and functional thrombin receptor.
Thrombin has numerous effects on a variety of cells outside of platelets. For example, thrombin is mitogenic for smooth muscle and endothelial cells. Thrombin is also known to increase vascular permeability and to induce vasoconstriction. See, Malik, Semin. Thromb. Hemostasis. 12:184-196 (1986). Thrombin can also induce the production and release of several constituents from endothelial cells including platelet-derived growth factor (PDGF), prostacyclin, platelet-activating factor (PAF), tissue plasminogen activator and plasminogen activator inhibitor. Finally, thrombin is capable of promoting the adherence of platelets, neutrophils, monocytes and T cells. For review, see, Shuman, Ann. NY Acad. Sci. 485:228-239, (1986). All of these actions of thrombin are likely to be mediated by cellular thrombin receptors identical or nearly identical to the cloned thrombin receptor and suggest that thrombin may also play a central role in initiating inflammatory and cellular proliferative responses to vascular injury linking it with the coagulation and hemostasis cascades. While most of these responses to thrombin suggest such a linkage between hemostasis and vascular repair, this hypothesis remains to be tested. Agents which specifically effect the activation of thrombin receptor(s) within cells are ideally suited to this purpose.
Restenosis, the vascular hyperproliferative response to blood vessel wall injury induced by interventional procedures such as coronary angioplasty, may be stimulated by thrombin-induced cellular events as sites of injury either directly or indirectly. Cellular proliferation may be simulated indirectly by the release of potent growth factors from locally adherent platelets or by the action of thrombin on endothelial cells which could release PDGF upon stimulation. Smooth muscle cell proliferation within diseased vessels may also be stimulated directly by thrombin due to the high local concentration of thrombin generated at the vascular injury sites created by the active platelet-rich thrombus. Indeed, recent studies with the potent thrombin inhibitor hirudin suggest that thrombin plays such a role in the restenosis process, but it is not known for these studies whether thrombin's effect is direct or indirect. See, Sarembock, et al., Circulation, 84:232-243, (1992).
Although the cellular actions of thrombin have the potential for causing various pathological conditions, there are no known therapeutic agents which specifically block the cellular actions of thrombin. Recently, however, a functional thrombin receptor cDNA has been cloned and expressed from megakaryoblastic cells lines, and the presence of mRNA encoding this receptor has been demonstrated in human platelets and vascular endothelial cells. See, Vu, et al., Cell, 64:1057-1068 (1991). This development has created significant opportunities to develop highly specific agents which target the cellular thrombin receptor.
Close inspection of the predicted amino acid sequence of the thrombin receptor revealed a potential recognition and thrombin cleavage sequence within the 100-residue amino terminal domain of the receptor. Subsequent mutagenesis studies of the receptor has demonstrated that this cleavage site is required for thrombin-mediated receptor signaling through proteolytic cleavage at this site. See, Vu, et al., Nature, 353:674-677 (1991). These experiments confirmed previous suggestions that proteolysis of a putative thrombin receptor might be responsible for thrombin receptor activation but left unanswered how proteolysis mediates receptor signaling.
Two potential explanations for thrombin-induced signaling have been postulated. The first is that proteolytic removal of the 15-residue segment at the amino terminus of the receptor induces a conformational change in the receptor leading to receptor activation. Alternatively a specific "tethered ligand" sequence unmasked upon receptor proteolysis may directly interact with a "ligand binding site" within the body of the receptor leading to receptor activation. While both of these potential mechanisms are quite similar, if not semantic, it appears that the "tethered ligand" hypothesis is the more likely explanation for receptor signaling. Synthetic peptides which mimic the new amino acid sequence revealed upon receptor proteolysis function as full agonists of the platelet receptor even in the absence of proteolytic cleavage of the receptor. This suggests that the new amino acid sequence at the amino terminus of the receptor revealed upon cleavage of the receptor functions as a "tethered ligand" and interacts at a distal "binding site". These effects have been confirmed with the hamster receptor activated with the hamster "tethered ligand" peptide. See, Vouret-Craviari, et al., Mol. Biol. Cell. 3:95-102, (1992). Additional studies with agonist peptides have confirmed the similarity of putative thrombin receptors present in platelets, endothelial cells, fibroblasts and smooth muscle cells. See, Hung et al., J. Cell. Biol. 116:827-832, (1992) and Ngaiza and Jaffe, Biochem. Biophys. Res. Commun. 179:1656-1661 (1991).
Most research conducted to date on modulating the actions of thrombin have been directed toward non-specific inhibition of the catalytic activity of thrombin. These efforts have resulted in thrombin inhibitors which effect both the pro- and anti-coagulant actions of thrombin. For a review, see, Chesbro and Fuster, Circulation, 83:1815-1817 (1991). Certain investigators have also attempted to inhibit the cellular activities of thrombin by using polypeptides prepared from the sequence of thrombin. See, Carney and Glenn, International Patent Application Under the PCT, No. WO 88/03151, 5 May 1988. Another report has focused on the preparation of certain dipeptides and analogs which appear to inhibit thrombin-cellular activities without effecting the catalytic activity of thrombin. See, Ruda, et al., Biochem. Pharmacol. 39:373-381 (1990). Both of these approaches display a certain degree of specificity but are limited due to their lack of potent effects.
Structure-activity studies using the thrombin receptor agonist peptide sequences have also been reported. A pentapeptide sequence, Phe-Leu-Leu-Arg-Asn-OH, based on a portion of the agonist peptide minimum structure has been shown to be a weak antagonist of platelet thrombin receptors activated with either thrombin or thrombin receptor agonist peptides. See, Vassallo, et al., J. Biol. Chem. 267:6081-6085 (1992).
A different approach to receptor antagonism has been investigated by others who have raised antibodies against a peptide sequence within the amino-terminal domain of the thrombin receptor which appears to be a binding/recognition site for thrombin. Thee antibodies effectively and specifically block thrombin-induced responses in platelets, thereby acting as antagonists of the thrombin receptor. See, Hung et al., J. Clin. Invest. 89:1350-1353 (1992).
The lack of potency and/or specificity of the above described approaches to thrombin receptor antagonism limits their utility as thrombin receptor antagonists. Thus, highly potent and specific inhibitors of the thrombin receptor are the focus of this invention.